One of the most detrimental effects of ethanol exposure during nervous system development is depletion of neuronal cells. Ethanol may deplete neurons by several means, one of them is ethanol-induced cell death, which is the focus of this proposal. Ethanol exposure of primary neuronal cultures of cerebellar granule cells (CGC) produces cell death and this cell loss is similar to that observed in animal studies. A valuable property of this model is that both ethanol-sensitive and ethanol-resistant cultures can be obtained in two ways. First, as CGC mature in culture they become ethanol-resistant, called time-dependent ethanol resistance. Second, treating ethanol-sensitive CGC with either NMDA or growth factors makes cells ethanol-resistant, called neuroprotection-dependent ethanol resistance. Of considerable value, ethanol-sensitive and ethanol-resistant cells can be compared to identify molecular differences which may be linked to ethanol-induced cell death. Specific Aim 1 will determine whether ethanol causes both necrotic and apoptotic cell death in CGC cultures. Our previous studies determined that the nitric oxide-cGMP (NO-cGMP) pathway is essential for NMDA-mediated neuroprotection against ethanol-induced cell death in CGC cultures. Specific Aim 2 will study whether the pathway plays an essential role in both time-dependent and neuroprotection-dependent ethanol resistance. CGC cultures will be established from mutant mice which lack the NO-cGMP pathway, in order to evaluate the function of the pathway in ethanol neurotoxicity and resistance. This specific aim may establish that the NO-cGMP pathway is activated by diverse signals (NMDA, growth factors, and time) and it plays an essential role in ethanol resistance. Since Ca2+ has been linked to cell death, Specific Aim 3 will examine the role of Ca2+ in ethanol-induced cell death in CGC cultures. Both ethanol-sensitive and ethanol-resistant cultures will be compared to determine whether there are differences in the maintenance of intracellular Ca2+, which may be linked to ethanol-induced cell death. In summary, this proposal will use ethanol-sensitive and ethanol-resistant cultures to obtain information about the nature of ethanol-induced cell death (necrotic versus apoptotic), the functional role of the NO-cGMP pathway in protecting neurons against this cell death, and the role of Ca2+ in this cell death. These closely-linked specific aims will greatly enhance our knowledge about molecular mechanisms involved in one of ethanol's most detrimental effects, neuronal cell death.